JP2002272458A - Method for sandwich assay using specific antibody to canine bnp - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for immunoassay of canine BNP. SOLUTION: This method for sandwich immunoassay of canine BNP comprises combining two kinds of monospecific antibodies which recognize canine BNP and do not mutually obstruct bond to canine BNP. The method for sandwich immunoassay of canine BNP comprises combining two kinds of a monoclonal antibody recognizing a part from Asn at 135-position to Tyr at 140-position among an amino acid sequence of sequence number 1 (reference to the specification) with a monoclonal antibody recognizing a part from Cys at 118-position to Cys at 134-position.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] 1. Field of the Invention [0002] The present invention relates to a monospecific antibody recognizing canine BNP, and a method for sandwich immunoassay of canine BNP using the antibody.

[0002]

2. Description of the Related Art In 1988, Sudoh et al. Obtained atrial diuretic peptide (AtrialNatriuret) from pig brain.
ic Peptide; ANP), a peptide having a structure very similar to that of ANP, was isolated and identified.
e; BNP) ( Nature , 332 (61
59), 78-81 (1988). ). After that, Sai
To et al. have shown that BNP is also synthesized in pig heart and secreted into the blood ( Biochemical B
iophysical Research Commu
nations , 158 (2), 360-368.
(1989). ). Since then, BNP has also been isolated from other animal species. In 1989, Itoh et al. Reported from the rat atria ( Biochemical and Biop).
physical Research Communication
ations , (2) 161, 732-739 (198
9). ), Seilhamer et al. From dogs ( Bioc)
chemical and Biophysical R
essearch Communications , 16
5 (2), 650-658 (1989). ) BNP was isolated, and in 1990, Kambayashi et al. Isolated BNP from human atria ( FEBS Letters ,
259 (2), 341-345 (1990). ).

[0003] Through clinical studies to date, human BNP
Has been widely applied clinically as a function evaluation method that reflects cardiac function in heart disease. on the other hand,
Dogs are one of the most bred pet animals by humans, and are an animal species that has entered human life without changing from family members.However, the incidence of heart disease is later than that of humans, etc. It is extremely common in dogs. In Japan, breeding dogs are registered and managed, and hospitals are well established, mainly in cities.However, there is no simple method for evaluating the function of the heart, so it is difficult to detect heart disease early. It is difficult and timely treatment is not possible. Against this background, the clinical application of BNP measurement is considered to be of great significance in the diagnosis and treatment of canine heart disease, including improvement of QOL in dogs, as in humans. I have. The canine BNP immunoassay reported to date since the isolation of canine BNP was performed using canine BNP as an antigen and antiserum obtained by immunizing rabbits. However, since an antiserum that is not specific enough to enable an efficient sandwich immunoassay was used, no highly sensitive assay system that could be used for clinical application was provided.

[0004]

Although a polyclonal antibody against canine BNP has been obtained, a specific antibody capable of binding to canine BNP, which is necessary for constructing a highly sensitive sandwich measurement system, has not yet been obtained. The present invention relates to dog B
An object of the present invention is to provide a monospecific antibody having a high property at a specific site which enables efficient sandwich immunoassay of NP, and a sandwich immunoassay method using the antibody.

[0005]

Means for Solving the Problems The present inventors have conducted studies for the purpose of providing a highly sensitive method for measuring canine BNP, and as a result, have found the present invention and completed the present invention.

That is, the present invention relates to (1) the amino acid sequence represented by SEQ ID NO: 1
A monospecific antibody recognizing canine BNP consisting of Tyr at position 140 from Ser at position 140 and usable for canine BNP sandwich immunoassay; (2) an amino acid sequence represented by SEQ ID NO: 1; (B) Cys at position 134 to Cys at position 134; or (c) Tyr at position 140 from Asn at position 135; (3) Recognizing the Tyr site at position 140 from Asn at position 135 in the amino acid sequence of SEQ ID NO: 1 (1)
(4) an antibody according to (3), which is a monoclonal antibody; (5) a mouse hybridoma dBC107 (FERM);
(18) the antibody of (4), which is produced by P-18005); (6) 118 of the amino acid sequences of SEQ ID NO: 1
The antibody according to (1), which recognizes the Cys site at position 134 from the Cys at position 134; (7) the antibody according to (6), which is a monoclonal antibody; (8) a mouse hybridoma dBR103 (FERM)
(18) an antibody according to (7), which is produced by P-18004); (9) a mouse hybridoma dBC107 (FERMP) that produces a monoclonal antibody against canine BNP.
(18005); (10) mouse hybridoma dBR103 (FERM) producing a monoclonal antibody against canine BNP
P-18004); (11) a sandwich immunoassay in which two antibodies selected from the antibodies described in (1) and (2) and which do not inhibit binding to dog BNP are combined; (12) Cys at position 118 A monospecific antibody that recognizes Cys at positions 134 through 134 and 1 from Asn at position 135
(11) the immunoassay according to (11), wherein a monospecific antibody recognizing Tyr at position 40 is combined; (13) a monoclonal antibody recognizing Cys from position 118 to position 134 and 140 from position 135 Asn. (11) the immunoassay according to (11), in which a monoclonal antibody recognizing Tyr at the position is combined; (14) the hybridoma dBC107 (FERMP)
-18005) and hybridoma dBR103 (F
(13) an immunoassay method according to (13), which comprises combining a monoclonal antibody produced by ERM P-18004); and (15) a dog containing the antibody according to any of (1) to (8). BNP measurement kit;

[0007] In the present invention, "monospecific antibody" means an antibody that recognizes a single epitope. Such antibodies include antibodies obtained by using a polypeptide containing an epitope of interest as an immunogen and antibodies obtained by affinity purification of only an antibody component that recognizes only a single epitope. For example, monoclonal antibodies can be mentioned. Accordingly, an antibody obtained by using a polypeptide having a plurality of epitopes as an immunogen, for example, a polyclonal antibody obtained by using a whole protein having a physiological activity as an immunogen, is a `` monospecific antibody '' of the present invention. Is not included. "Canine BNP"
Means a polypeptide consisting of 32 amino acid residues from Ser at position 109 to Tyr at position 140 in the amino acid sequence described in SEQ ID NO: 1. Also, "dog BNP"
Corresponds to position 118 and 1 of the amino acid sequence described in SEQ ID NO: 1.
Cys at position 34 may form an SS bond and become a cystine, thereby forming a cyclic structure. In such a structure, Ser from position 109 of SEQ ID NO: 1 to 11
The site consisting of 9 amino acid residues of Gly at position 7 is “N-terminal”, the site consisting of 17 amino acid residues from Cys at position 118 to Cys at position 134 is “ring”, and An at position 135
The site consisting of 6 amino acid residues of Tyr at position 140 from s is referred to as “C-terminal”.

[0008] "Can be used for sandwich immunoassay" refers to a monospecific antibody that recognizes different epitopes, and inhibits the binding of the antibody to a target analyte, for example, canine BNP. Means that they can be combined without any modification. In the present invention, “mouse hybridoma dBC107” is a hybridoma that produces a monoclonal antibody that recognizes the C-terminal sequence of canine BNP, and the hybridoma is 1-1-1 Higashi, Tsukuba, Ibaraki, Japan
Accession No. 1 to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST) on August 29, 2000, accession number:
Deposited as FERM P-18005. In the present invention, the “mouse hybridoma dBR103” is a hybridoma that produces a monoclonal antibody that recognizes a ring of canine BNP, and the hybridoma is a 1-1-1 east Higashi 1-chome, Tsukuba, Ibaraki Prefecture, Japan. Research Institute Patent Organism Depositary Center
Accession number: FERM P-18 on August 29, 2
No. 004.

[0009] The "sandwich immunoassay method in which two kinds of monospecific antibodies which do not mutually inhibit the binding to dog BNP" is described as "two kinds of epitopes which recognize dog BNP monospecifically and have different epitopes." A sandwich immunoassay using an antibody, wherein the antibody is an immunoassay in which one of the antibodies is capable of binding to dog BNP without being inhibited by the other. Such 2
Examples of combinations of monospecific antibodies include, for example,
Monospecific antibodies at the N-terminus and ring, monospecific antibodies at the N-terminus and C-terminus, and combinations of monospecific antibodies at the ring and C-terminus are preferred. This is a combination of a ring and an antibody that monospecifically recognizes the C-terminus. As the two types of monospecific antibodies used in the present measurement method, any antibody may be used as long as it recognizes a single epitope, preferably a monoclonal antibody, and more preferably a mouse hybridoma. dBC107 and mouse hybridoma dBR
103 is a monoclonal antibody produced by

[0010] The "assay kit" contains the monospecific antibody of the present invention, and preferably contains the two monospecific antibodies of the present invention. One of the two antibodies used may be immobilized on a carrier. The solid phase of the antibody on the carrier may be direct or indirect. Examples of the indirect solid-phase method include an antibody for the antibody to be used (however, one that does not inhibit the binding to the measurement object), avidin-
Examples of the method include a combination of biotin and an antigen / antibody different from the measurement target. Further, a solid support may be included in the measurement kit. Further, the other antibody may be labeled. The binding of the label to the antibody may be direct or indirect. Examples of the label include a radioisotope, an enzyme, and a fluorescent substance. Preferably, they are ¹²⁵ I, peroxidase, alkaline phosphatase and luminol. The indirect labeling of the antibody may be a combination of an antibody to the antibody to be used to which the label is bound (however, it does not inhibit the binding to the measurement target), avidin-biotin, an antigen antibody different from the measurement target, and the like. A method is illustrated. The kit can further include a calibration curve, a standard reagent, instructions for handling the standard reagent, and the like.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention will be described. To produce monospecific antibodies against immune dog BNP, first, immunizing an animal with the appropriate antigen. In particular, when an antibody is prepared in consideration of the binding site between the antibody and dog BNP, a suitable immunogen capable of producing the antibody using a peptide containing only the binding site is prepared, and Immunize. Animals used for immunization include, for example, mice, rats, rabbits, goats and the like. As the antigen, canine BNP itself or a peptide fragment of any part thereof can be used, and preferably a C-terminal or ring peptide fragment. Peptide fragments are chemically synthesized by a peptide synthesizer based on amino acid sequence information and can be used as an immunogen. The immunogen can be used as is,
It can also be used in a conjugate in combination with a carrier protein. As a carrier protein,
For example, macromolecules such as bovine serum albumin (BSA), hemocyanin, and bovine thioglobulin (BTG) can be mentioned, and BSA is preferable. In order to prepare a conjugate, the peptide and the carrier protein may be combined using a crosslinking reagent. As such a crosslinking reagent, a maleimide compound that is a bifunctional crosslinking reagent having a succinimidyl group and a maleimide group can be used, and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), m- Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl 4- (p-maleimidophenyl) butyrate (SM
PB), and Sulfo-MBS, Sulfo-S
MCC etc. are illustrated. The succinimidyl group of these crosslinking reagents reacts with the primary amine and further reacts with the thiol-reactive maleimide, forming a covalent bond with the thiol group of the cysteine residue and crosslinking. To enhance the immune response by the immunogen, for example, Freund's complete adjuvant (CFA) and incomplete adjuvant (IFA) may be administered.

Polyclonal antibodies can be obtained, for example, by the method of Lane et al. ( Antibodies ; Alaborator).
y Manual, Cold Spring Harb
er Laboratory Press, New Y
ork (1989). ) And the like. Intraperitoneally the emulsion emulsified in a suitable adjuvant such as Freund's complete adjuvant,
The immunized mammals can be obtained by subcutaneous or intravenous administration and multiple administrations over several weeks to obtain immunized mammals, and serum can be collected from those mammals and purified. The antibody titer in the blood was determined by labeling dog BNP with iodine-125.
It can be examined by a competitive RIA using ¹²⁵ I-Y ₀ -dog BNP-32 as a tracer. When it is desired to remove an antibody that binds to a site other than the target site of canine BNP, the carrier can be prepared by immobilizing a peptide at the target site on a solid phase and affinity-purifying by a known method.

[0013] Anti-canine BNP mouse monoclonal antibody
Preparation As for monoclonal antibodies, monoclonal antibody-producing hybridomas can be obtained by collecting spleens or lymph nodes from those mammals and fusing antibody-producing cells obtained therefrom with myeloma (myeloma) cells. . Cell fusion can be performed by a known method, for example, the method of Kohler and Milstein ( Nat
ure , 256 , 495-497 (1975). ). Mice are immunized according to the method described above to generate specific antibodies that recognize canine BNP. Confirm that the blood antibody titer is sufficiently high, and collect blood or spleen cells. A hybridoma that produces a monoclonal antibody, particularly a monoclonal antibody that recognizes the C-terminus or ring, can be produced by fusing the spleen cells thus isolated with myeloma cells. The spleen cells are derived from the animal, preferably a mouse, immunized above. Myeloma cells are of mammalian origin,
Preferably, it is a mouse myeloma cell. For cell fusion, polyethylene glycol or the like can be used. The desired hybridoma can be selected by screening and cloning the hybridoma obtained by the fusion. To prepare a monoclonal antibody, the hybridoma obtained above is cultured in vitro or in vivo. Preferably, it is cultured in vivo. For example, the hybridoma is administered intraperitoneally to a mouse in order to produce ascites containing a mouse monoclonal. Monoclonal antibodies can be readily purified from the produced ascites by methods known to those skilled in the art.

[0014] The monospecific antibodies used in immunoassays the immunoassay, a monoclonal antibody capable of supplying stably desirable. Hereinafter, an example will be described using a monoclonal antibody. Immobilizing the antibody (the first monoclonal antibody) on a solid phase and incubating with the material containing the antigen, adding a labeled second monoclonal antibody, incubating the resulting mixture, and producing the mixture A sandwich immunoassay including a step of detecting a labeled labeled antigen-antibody complex is exemplified. In the immunoassay of the present invention, the sample and the immobilized first monoclonal antibody and labeled second monoclonal antibody may be simultaneously incubated.

The sandwich immunoassay of the present invention includes, as detection methods, sandwich radioimmunoassay (RIA) and sandwich enzyme immunoassay (EIA).
Method, sandwich fluorescent immunoassay (FIA method), sandwich luminescence immunoassay (CLIA method), sandwich luminescent enzyme immunoassay (CLEIA method), and immunochromatography based on the sandwich method. Can be applied. For quantification, RIA
And the EIA method are preferred. The first monospecific antibody of the present invention can be immobilized on a carrier such as a microtiter plate, a bead, a tube, a membrane, a filter paper, or a plastic cup, and polyethylene beads are particularly preferably used. The sample to be measured can be a sample containing dog BNP, such as dog plasma, serum, blood, urine, and the like. The second monoclonal antibody of the present invention is a radioisotope,
In the case of an enzyme, a fluorescent substance, a luminescent substance, or a visual determination method that can be visually determined, the labeling can be performed using gold colloid or colored latex. The radioisotope used for labeling is ¹⁴ C, ³ H, ³² P, ¹²⁵ I, ¹³¹ I, etc., and ¹²⁵ I is particularly preferably used. These can be bound to the monoclonal antibody by the chloramine T method, the peroxidase method, the iodogen method, the bolt hunter method, or the like. Examples of the enzyme that can be used for labeling include β-galactosidase (βGAL), alkaline phosphatase (ALP), horseradish peroxidase (HRP), and the like. These are described in Nakane method, Ishikawa et al. Method (Medical Shoin; Enzyme immunoassay, 3rd edition, 75-127, (198)
7). ) And the like. Fluorescein,
Fluorescamine, fluorescein isothiocyanate,
And tetramethylrhodamine isothiocyanate. Luminescent substances used for labeling include luciferin, luminol derivatives, and acridinium esters.
In a simple measurement method or the like, gold colloid or colored latex may be used.

According to a preferred embodiment, a canine BNP sandwich RIA method can be performed. In the dog BNP sandwich RIA method, specifically, a standard dog BNP solution or sample is mixed with beads to which a first monoclonal antibody has been immobilized, and mixed at 4 ° C to 45 ° C, preferably 25 ° C to 37 ° C. Incubate for 1 to 4 hours, preferably 2 hours (first reaction). After washing, a solution containing, for example, a ¹²⁵ I-labeled second monoclonal antibody is added, and the solution is added at 4 ° C.
From 4 to 45 ° C, preferably from 25 to 37 ° C, from 1 to 4
Incubate for a period of time, preferably 2 hours, to form an antibody / dog BNP / antibody complex on the beads (second reaction). After washing, the amount of canine BNP can be measured by detecting the radioactivity of the antigen-antibody complex bound to the beads using a gamma counter or the like. According to another preferred embodiment, a canine BNP sandwich EIA method can be performed.
The dog BNP sandwich EIA method is, specifically, a standard dog BNP solution or a sample, a bead on which a first monoclonal antibody is immobilized is added, and the mixture is mixed at 4 ° C to 45 ° C.
Incubate at preferably 25 ° C. to 37 ° C. for 1 to 4 hours, preferably 2 hours (first reaction). After washing
Enzyme labels such as horseradish peroxidase (HR
Add a solution containing a second monoclonal antibody labeled with P) and add 4 ° C to 45 ° C, preferably 25 ° C to 37 ° C.
And incubate for 1 to 4 hours, preferably 2 hours,
An immune complex consisting of the antibody-dog BNP-antibody is formed on the beads (second reaction). The enzymatic activity on the beads is measured colorimetrically via a substrate specific for the enzyme, for example, tetramethylbenztin (TMB) if the labeling enzyme is HRP, whereby the captured dog on the beads is measured. The amount of BNP can be measured. The colorimetric determination can be performed with a usual spectrophotometer or the like.

[0017]

The present invention is further described by the following examples. Example 1 Setting of Competitive RIA Method for Canine BNP Anti-human BNP monoclonal antibody KY-h which recognizes the ring site of human BNP and has cross-linking ability with canine BNP
BNP-II (Japanese Patent Application No. 2-99623) and ¹²⁵ I-
By using Y ₀ -human BNP (1-32), a competitive RIA was constructed for canine BNP measurement. The hybridoma KY-hBNP-II producing the monoclonal antibody KY-hBNP-II was introduced in 1990 (1990).
From April 11th, the Mouse Hybridoma KY-h was registered with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology at 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture.
BNP-II, accession number: FERM BP-2863, deposited under the Budapest Treaty. RIA
Contains 0.01M PBS as an assay buffer, pH
7.4 (1 mM EDTA · 2Na, 0.01% (W /
V) NaN ₃ , containing 0.2% (W / V) BSA)
It was used. ¹²⁵ I-Y ₀ -human BNP was obtained by the chloramine T method ( Biochemical and Biophysi).
cal Research Communicatio
ns , 124 (3), 815-821 (1984). )
Prepared by A standard curve for human BNP and dog BNP was prepared by the following method. First, 100 [mu] L of BNP standard solutions of various concentrations in polystyrene tubes, ¹²⁵
100 μL of I-Y ₀ -human BNP (40,000 cp)
m), 100 μL of the monoclonal solution and 200 μL of the assay buffer were dispensed, stirred, and then stirred at 4 ° C. for 20 minutes.
Incubated for hours. 2.5% (W /
V) 100 μL of γ-globulin and 25% (W /
V) 100 μL of PEG-6000 was added, and the mixture was stirred at 4 ° C. for 10 minutes, centrifuged at 3,000 rpm for 20 minutes, and the radioactivity of the obtained precipitate was measured with a gamma counter.

The relationship between the radioactivity and various concentrations in the standard solution of human BNP and dog BNP is shown in FIG. 1 as a standard curve. In the figure, the horizontal axis represents various BNP concentrations, and the vertical axis represents the ratio (B / B ₀ ) between the radioactivity (B) at various BNP concentrations and the radioactivity (B ₀ ) at zero BNP concentration. As a result, the reaction of the monoclonal antibody KY-hBNP-II that recognizes the ring with the labeled antigen was competed by canine BNP, and a volume-dependent competition curve having a minimum detection limit almost equivalent to that of human BNP was drawn. Therefore, further optimization of the reaction system was performed. FIG. 2 shows a standard curve showing the relationship between various concentrations and radioactivity in a standard solution of canine BNP, and the measurement results of three types of plasma samples collected from normal dogs. As a result, the minimum detection limit of canine BNP was about 600 pg / mL, and no competition occurred in plasma samples collected from normal dogs.
Therefore, clinical application of canine BNP measurement required the construction of a more sensitive assay such as a sandwich immunoassay.

Example 2 Preparation of Monoclonal Antibody (1) Monoclonal Antibody d Recognizing BNP Ring
Preparation of BR103 About 2 mg of canine BNP prepared by peptide synthesizer
9-10 mg of (10-26) using carboximide
Of beef thyroglobulin (Sigma). The above compound containing about 20 μg of dog BNP (10-26) emulsified in Freund's complete adjuvant was administered subcutaneously and intraperitoneally to the back of BALB / c mice at intervals of 2 to 3 weeks. Was. Since the antibody titer was increased in 5 out of 6 mice, spleen cells for monoclonal antibody production were excised from one mouse showing higher reactivity and used for cell fusion. Fusion of spleen cells with mouse myeloma cells X63-Ag8.653 was performed by Muk
Oyama et al.'s method ( Hypertension , 1).
2, 117-121 (1988). A). After the cell fusion, all the fusion strains that showed a positive antibody reaction were cloned, and the clone having the strongest reactivity was selected.
The monoclonal antibody thus established was identified as dBR10
No. 3. The resulting hybridomas were selected based on their ability to produce antibodies, cloned by limiting dilution, and
ALB / c mice were grown intraperitoneally. The isotype of the monoclonal antibody was determined by the Ouchterlony method using a mouse monoclonal typing kit (manufactured by Miles). The affinity constant (Ka (M ^-1 )) is expressed by Sca
tchard et al., Scatchard blot (Ann.
N. Y. Acad. Sci. , 51, 660 (194)
9). ). dBR103 has an affinity of 3.0 × 10 ⁸ M ⁻¹ for canine BNP. However, the cross-reactivity with human BNP and human ANP was 0.01% in each case.

(2) Preparation of monoclonal antibody dBC107 recognizing the C-terminus of BNP Monoclonal antibody dC107 recognizing the C-terminus using dog BNP (27-32) as an antigen in the same manner as in the above (1).
BC107 was produced. After the cell fusion, all the fusion strains that showed a positive antibody reaction were cloned, and the clone having the strongest reactivity was selected. The monoclonal antibody thus established was named dBC107. dBC1
07 was cultured in the same manner as in the above (1), and Ka was calculated. dBC107 was 1.7 × 10 ¹⁰ against dog BNP
It has an affinity of M- ¹ . However, the cross-reactivity with human BNP and human ANP was 0.0
1%.

(3) Preparation of antiserum to dog BNP For comparative study of the monoclonal antibodies obtained in the above (1) and (2), antiserum to dog BNP was prepared. Immunization was performed using dog BNP as an immunogen and five rabbits as immunized animals. The immunogen was immunized a total of five times at 2-3 week intervals, and the antibody titer was measured at each time. Antiserum was prepared according to a conventional method. Table 1 shows the results. However, "-" indicates that blood collection was not performed.
Antisera-4 out of the five rabbits showed a high antibody titer of 83,000 at the time of the fourth immunization. Therefore, the antiserum used for the study was selected as a subject to be collected, and blood was collected to obtain the antiserum. Prepared. In addition, the affinity constants (Ka) of various antibodies used in this study and human BNP and human AN
The cross reactivity with P is shown in Table 2.

[Table 1]

[Table 2]

Example 3 (1) Analysis of Complex of Canine BNP and Antibody by Gel Filtration Chromatography The complex formed by canine BNP and antibody was evaluated by gel filtration. As antibodies, monoclonal antibodies dBC107 and dBR103 and rabbit anti-canine BNP antiserum Antisera-4 were used. ¹²⁵ I-labeled form of dog BNP ( ¹²⁵ I-Y ₀ -dog BN
P) was prepared in the same manner as labeled human BNP by the chloramine T method, and the chromatographic buffer (0.2% B
(Including SA). As a reagent, assay buffer 0.01M PBS, pH 7.4 (1 mM
EDTA · 2Na, 0.01% (W / V) Na
N ₃ , 0.2% (W / V) BSA) and chromatography buffer 10 mM Tris-NaCl,
A pH of 7.0 (containing 0.15 M NaCl) was used. All chromatographic operations were performed at room temperature. First, dB adjusted to an appropriate concentration in a polystyrene tube
100 μL of ¹²⁵ I-Y ₀ -dog BNP solution and 200 μL of assay buffer were added to 100 μL of the C107 antibody solution, 100 μL of the dB107 / dBR103 antibody solution, and 100 μL of the antibody solution, and stirred each sample. Afterwards, it was incubated overnight at room temperature. Each of the obtained reaction solutions was fractionated by gel filtration chromatography using a Superose HR12 column (manufactured by Pharmacia Biotech). Each sample was applied to a Superose HR12 column equilibrated with the chromatography buffer, and eluted at a flow rate of 0.5 mL / min. All eluate from the column was 0.01 M PBS, pH 7.4 (2%
0.5 mL was fractionated into a polypropylene tube containing 100 μL of BSA (including BSA), and the radiation dose in the tube was measured with a γ-counter. The result is shown in FIG. ¹²⁵ I-Y ₀ - only those dogs BNP (□), ^{12 5}
I-Y ₀ - combination of canine BNP and dBC107 is ^{(◇), 125 I-Y} 0 - combination of canine BNP and dBC107 and dBR103 showed elution pattern (●). ¹²⁵ I-Y ₀ -canine BNP was used for ¹²⁵ I-
The complex in which Y ₀ -canine BNP and antibody were bound 1: 1 was 2
Fourth, the sandwich complex bound 1: 2 was 22
Each eluted in the first run.

Antisera prepared at an appropriate concentration
100 μL of the 4 antibody solution, 100 μL of the dBC107 / Antisera-4 antibody solution, and 100 μL of the ¹²⁵ I-Y ₀ -dog BNP solution,
L and 200 μL of assay buffer were added and each sample was stirred and then incubated overnight at room temperature. Each of the obtained reaction solutions was fractionated by gel filtration chromatography using a Superose HR12 column (manufactured by Pharmacia Biotech). Each sample was applied to a Superose HR12 column equilibrated with the chromatography buffer in the same manner as described above, and eluted at a flow rate of 0.5 mL / min. Perform gel filtration chromatography and use 0.01M PB
S, pH 7.4 (containing 2% BSA) 0.5 mL in a polypropylene tube containing 100 μL,
The radiation dose in the tube was measured with a gamma counter. The results are shown in FIG. ¹²⁵ I-Y ₀ -dog BN
The P only ones ^{(□), 12 5 I-} Y 0 - Dog BNP and An
The combination of tisera-4 (◇) and the combination of ¹²⁵ I-Y ₀ -dog BNP with Antisera-4 and dBC107 (−４) showed elution patterns. ¹²⁵ I-Y ₀
- Dog BNP is a 40 knots, ¹²⁵ I-Y ₀ - Dog BNP
The complex in which the antibody and the antibody were bound 1: 1 was the 24th one,
The sandwich complex bound by the above was eluted on the 22nd line, respectively.

(2) Investigation of the formation of a sandwich complex by combining antibodies In general, two or more antibody binding sites (epitope)
Since an antiserum obtained by immunizing a compound having the above-mentioned formula (1) produces antibodies against the respective epitopes, a sandwich immune reaction is possible even when the same antiserum is used for the solid phase and the label. If the object to be measured is a peptide having a certain size, the number of epitopes is usually rarely 2 or less, and a macromolecule is often formed as a result of the reaction with the antiserum. Once such a macromolecule forms a complex, dissociation hardly occurs due to heating, washing, and the like, and it is often preferable to construct a measurement system. Since human BNP can produce antibodies at the N-terminus, ring site, and C-terminus, it is supposed that at least three or more epitopes are present. In fact, since sandwich measurements are possible, two or more antibodies may bind simultaneously. Canine BNP has a different amino acid sequence from human BNP, and its higher-order structure cannot be inferred. However, it can be estimated that a sandwich measurement system can be constructed by selecting at least a high-titer antiserum.

In order to increase the sensitivity of the sandwich measurement system, it is necessary that as many antigen molecules as possible react and no unreacted antigen molecules remain in the reaction solution. Rabbit anti-dog BNP antiserum Antisera-4 selected this time
Indicates that the antibody titer at the time of blood collection was 83.0 as shown in Table 1.
00 fold higher affinity constant (Ka) was good with ^{2 × 10 -9, 125 I-} Y 0 - According to gel filtration chromatography analysis in combination with dog BNP, ¹²⁵ I-Y ₀ - Dog BNP
Is recovered from the elution position of the complex with the antibody, but ¹²⁵ I-
Almost no recovery was obtained from the single elution position of Y ₀ -dog BNP. This indicates that antiserum Antisera-4
Can bind most of the ¹²⁵ I-Y ₀ -canine BNP molecules present in the reaction system. From this viewpoint, this antiserum is an antiserum suitable for a canine BNP immunoassay line. However, Antisera-
The reaction solution of 4 with ¹²⁵ I-Y ₀ -dog BNP was mostly 2
Since it is recovered from the fourth elution position, ¹²⁵
I-Y ₀ -is a complex in which one molecule of antibody is bound to dog BNP, and ¹²⁵ I-Y ₀ -is recovered from the elution position of the 22nd line.
The complex in which two molecules of the antibody bound to dog BNP was about 20% of the total complex formed. This ratio did not change significantly when the concentration of antiserum was changed.

When an antiserum against canine BNP is prepared by a usual preparation method, antibodies are prepared against a plurality of epitopes on canine BNP. However, if an antiserum is simply selected using only the antibody titer as an index, a sandwich reaction does not occur in most of the dog BNP molecules to be measured, and only a complex in which only one antibody is bound is formed. That is, even if a sandwich immunoassay is constructed using Antisera-4 alone, the antigen cannot be effectively incorporated into the assay system, and it is difficult to increase the sensitivity. There are several possible causes for the majority of the complex to which only one antibody binds, but if the ratio does not change in a concentration-dependent manner, this is not due to the shortage of the antibody in the reaction solution. It is highly probable that antibodies to any of the epitopes are preferentially produced and that binding of the antibodies to dog BNP inhibits the binding of other antibodies.

On the other hand, a monoclonal antibody recognizing the C-terminus
With the body dBC107¹²⁵I-Y₀-Reaction solution with dog BNP
From the gel filtration chromatography analysis, the 24th elution
Because it was recovered from the location, dBC107¹²⁵
I-Y₀-Binds to a majority of canine BNP to form a complex
Canine BN as in Antisera-4
It is clear that the antibody can be used for P immunoassay
(FIG. 3). Therefore, ¹²⁵I-Y₀-Canine BNP and An
Antisera-4 in combination with tisera-4
Ka value 2 × 10^-TenDBC107 anti-
After adding the reaction mixture, the reaction solution was subjected to gel filtration chromatography.
From the Raffy analysis, it was collected from the elution position of the 22nd,
All dog BNP formed a sandwich complex (primary
4). Difficult to deduce the three-dimensional structure of canine BNP molecule
Although it binds only to the C-terminus, antisera-4
With a BC107 antibody with a higher Ka value¹²⁵I-Y₀−
The reaction of canine BNP can interfere with other antibodies.
Inhibits the reaction of the antibody that was previously used, allowing the remaining antibody to bind
It is considered to have been.

Next, when a monoclonal antibody dBR103 antibody recognizing a canine BNP ring was added to the combination of the dBC107 antibody and ¹²⁵ I-Y ₀ -canine BNP,
From the gel filtration chromatography analysis of the reaction solution, 2
Recovered from the fourth elution position, dog BNP formed a sandwich complex (FIG. 3). This result was obtained by constructing a measurement system by combining antibodies specifically recognizing the C-terminal and the ring.
It shows that most of the ¹²⁵ I-Y ₀ -dog BNP molecules can be in a sandwich complex,
It was confirmed that the combination was an antibody suitable for a sandwich immunoassay for P-sensitive measurement.

(3) Sandwich RIA method A sandwich RIA system for canine BNP was established using a combination of both antibodies dBC107 and dBR103. 0.01MPB as assay buffer as reagent
S, pH 7.4 (1 mM EDTA · 2Na, 0.01
% (W / V) NaN ₃ , 0.2% (W / V) BSA
) As a washing solution, 0.01M PB, pH7.
2 (including 0.02% (W / V) Tween 20) was used. First, antibody solid-phase beads were prepared. According to a conventional method, immunobeads (D = 6.4) were added to a dBC107 antibody solution adjusted to a concentration of 100 μg / mL with an assay buffer.
mm) was left at room temperature for 2 hours to immobilize the antibody. Then, it was blocked using a 4-fold diluted Block Ace solution to obtain antibody solid phase beads. Also, according to the usual method, d
To label the BR103 antibody with the radioactive isotope ¹²⁵ I,
Introducing ^{1 25} I by the chloramine T method, and the labeled antibody.
In a reaction tube, add 100 standard dog BNP solution or sample
Dispense μL and add labeled antibody (300,000c) to each tube.
pm) 200 μL and one antibody solid phase bead were added and gently stirred. The reaction tube is sealed, and
After standing for 8 hours, the beads were washed four times with a washing solution, and the radioactivity of the beads was measured with a gamma counter. A standard curve was prepared from the relationship between dog BNP concentration in the standard solution and radioactivity. The result is shown in FIG. dBC1
The minimum detection limit of the sandwich RIA method using both antibodies 07 and 103 is 10 pg / mL or less, and is approximately several tens of pg / mL in healthy dog BNP ( Journalof Veterinarnar) in plasma.
y Medical Science , 61, 523-
529 (1999). ) Was considered to be a measurement system with sufficient sensitivity to measure

[0030]

As described above, canine BNP consists of the same 32 amino acid residues as human BNP, but has a different amino acid sequence (FIG. 5). Therefore, the three-dimensional structure of canine BNP was
It is impossible to catch the same as in the case of P. By the way, an antiserum obtained by using as an immunogen a molecule having two or more antibody binding sites (epitope) on a molecule uses the same antiserum as a solid phase and a label because an antibody against each epitope is produced. It is also believed that a sandwich composite can be formed. Thus, a rabbit antiserum was prepared according to a conventional method, and an attempt was made to construct a sandwich immunoassay for dog BNP. As a result, it was confirmed that a plurality of sandwich-capable epitopes were present on dog BNP. However, many dog BNPs in the reaction did not form a sandwich complex. From this, the antibody that has been selected from the antibody titer which is a conventional index is the canine BNP.
It was found that this method was not suitable for the construction of a sandwich immunoassay method. In order to solve this problem, a specific epitope was determined from the time of preparing the antibody, and an attempt was made to prepare a specific antibody that binds only to the epitope. As a result, it was clarified that most of the canine BNP present in the reaction system formed a sandwich complex by combining the specific antibody of the present invention, particularly the specific antibody for the C-terminus and the antibody for the ring site. Therefore, the present invention provides a highly sensitive immunoassay method for canine BNP, which was conventionally difficult.

[0031]

[Sequence List] SEQUENCE LISTING <110> Shionogi & Co., Ltd. <120> A sandwich immunoassay using specific antibodies against dog BNP <130> 01P00104 <150> JP2000-299300 <151> 2000-09-29 <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 140 <212> PRT <213> Canis familiaris <300> <301> Seilhamer, Jeffrey J. Arfsten, Ann Miller, Judy A. Lundquist, Penny Scarborough, Robert M Lewicki, John A. Porter, J. Gordon <302> Human and canine gene homologs of porcine brain natriuretic peptide <303> Biochem. Biophys. Res.Commun. <304> 165 <305> 2 <306> 650-658 < 307> 1989-12-15 <308> P16859 <309> 1990-08-01 <400> 1 Met Glu Pro Cys Ala Ala Leu Pro Arg Ala Leu Leu Leu Leu Leu Phe 1 5 10 15 Leu His Leu Ser Pro Leu Gly Gly Arg Pro His Pro Leu Gly Gly Arg 20 25 30 Ser Pro Ala Ser Glu Ala Ser Glu Ala Ser Glu Ala Ser Gly Leu Trp 35 40 45 Ala Val Gln Glu Leu Leu Gly Arg Leu Lys Asp Ala Val Ser Glu Leu 50 55 60 Gln Ala Glu Gln Leu Ala Leu Glu Pro Leu His Arg Ser His Ser Pro 65 70 75 80 Ala Glu Alu Pro Glu Ala Gly G ly Thr Pro Arg Gly Val Leu Ala Pro 85 90 95 His Asp Ser Val Leu Gln Ala Leu Arg Arg Leu Arg Ser Pro Lys Met 100 105 110 Met His Lys Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly Ser 115 120 125 Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Lys Tyr 130 135 140

[Brief description of the drawings]

FIG. 1 shows a competitive R using a monoclonal antibody (KY-hBNP-II) that recognizes the ring structure of human BNP.
Human BNP (1-32) and canine BNP of the IA method
It shows the standard curve for (1-32).

FIG. 2 shows a competitive system R using a monoclonal antibody (KY-hBNP-II) that recognizes the ring structure of human BNP.
Fig. 3 shows a profile of reactivity of the IA method to plasma derived from normal dogs.

FIG. 3 shows a reaction product of a monoclonal antibody recognizing the C-terminus of canine BNP (dBC107) and labeled canine BNP (◇), a monoclonal antibody recognizing a ring structure of canine BNP (dBR103) and a monoclonal antibody dB
FIG. 2 shows elution profiles of a reaction product of C107 and labeled dog BNP (●) and labeled BNP alone (□) by gel filtration chromatography.

FIG. 4. Rabbit antiserum against dog BNP (Anti
sera-4) and a labeled product of canine BNP (、), a reaction product of a monoclonal antibody recognizing the C-terminus of canine BNP (dBC107) and Antisera-4 (●), and a labeled BNP alone (□) 1 shows an elution profile of gel filtration chromatography.

FIG. 5 shows the amino acid sequences of BNP of various animals.

FIG. 6 shows a canine BNP using a monoclonal antibody recognizing the C-terminus of canine BNP (dBC107) and a monoclonal antibody recognizing a ring structure (dBR103).
FIG. 3 shows the results (standard curves) of the measurement of various canine BNP standard solutions by the sandwich RIA method.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12P 21/08 C12N 5/00 B (72) Inventor Hideki Ota 5-chome Sagisu, Fukushima-ku, Osaka-shi, Osaka No. 12-4 Shionogi & Co., Ltd. F term (reference) 4B024 AA11 BA43 GA05 HA15 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA91 AB02 CA25 CA46 4H045 AA11 AA20 AA30 CA40 DA76 EA50

Claims (15)

[Claims] 1. The amino acid sequence of SEQ ID NO: 1
Dog B consisting of Ser at position 109 to Tyr at position 140
Monospecific antibodies that recognize NP and can be used in sandwich immunoassays for dog BNP. 2. An amino acid sequence represented by SEQ ID NO: 1. (a) Gly from Ser to 117th position to 117th position; (b) Cys from 118th position to 134th position; or (c) 135th position from Asn. The antibody according to claim 1, which recognizes any one site of Tyr at position 140. 3. The amino acid sequence of SEQ ID NO: 1
The antibody according to claim 1, which recognizes Tyr at position 140 from Asn at position 135. 4. The antibody according to claim 3, which is a monoclonal antibody. 5. The mouse hybridoma dBC107 (F
The antibody according to claim 4, which is produced by ERM P-18005). 6. The amino acid sequence of SEQ ID NO: 1
The antibody according to claim 1, which recognizes Cys at position 134 from Cys at position 118. 7. The antibody according to claim 6, which is a monoclonal antibody. 8. The mouse hybridoma dBR103 (F
The antibody according to claim 7, which is produced by ERM P-18004). 9. A mouse hybridoma dBC107 (FER) producing a monoclonal antibody against canine BNP.
MP-18005). 10. A mouse hybridoma dBR103 (FE) producing a monoclonal antibody against canine BNP.
RM P-18004). 11. A sandwich immunoassay in which two monospecific antibodies selected from the antibodies according to claim 1 and 2 that do not inhibit binding to dog BNP are combined. 12. Cys at position 118 to Cy at position 134
and a specific antibody that recognizes Asn at position 135
The immunoassay according to claim 11, wherein a monospecific antibody that recognizes Tyr at positions 140 to 140 is combined. 13. Cys at position 118 to Cy at position 134
antibody recognizing s and As at position 135
The method according to claim 11, wherein a monoclonal antibody recognizing Tyr at position 140 from n is combined. 14. The hybridoma dBC107 (FER)
MP-18005) and the hybridoma dBR10
14. The method according to claim 13, wherein two kinds of monoclonal antibodies produced by FERM P-18004 are used. A kit for measuring canine BNP, comprising the antibody according to any one of claims 1 to 8.